Biotin-labeled red cell survival in pediatric patients with sickle cell disease who form antibodies of uncertain clinical significance

Patients of all ages with sickle cell disease (SCD) who are frequently transfused develop more auto- and alloantibodies against RBC antigens compared to the general population of transfused patients.[1] [2] In addition, chronically transfused children, compared to those who are infrequently transfused, are over three times more likely to develop warm autoantibodies (WAA),[3] which are often antibodies of uncertain  (clinical) significance (AUS). When patients also possess variant RH genotypes and form AUS, providing compatible blood is additionally challenging. These medical and laboratory findings are seen among patients with hemoglobinopathies and may delay the provision of blood.

The in vitro monocyte monolayer assay (MMA) test may be useful in some situations to predict in vivo red cell survival in the setting of AUS, but results are time-limited, and transfusions should be given soon after receiving test results.[4] Also, the MMA cannot predict the in vivo rate of clearance of a particular unit. Alternately, on an investigational basis, donor RBCs may be labeled with biotin prior to patient transfusion to assess and predict in vivo survival kinetics over the lifespan of the cells. However, an autologous study in healthy adults found that 14% developed antibodies to biotinylated RBCs (B-RBCs); and in some of these subjects, B-RBC clearance was accelerated.[5] Thus, B-RBC antibodies should be considered AUS.

A recently published study by Yee and colleagues focused on four adolescent and young adult patients on chronic transfusion therapy for SCD who developed AUS during an ongoing trial of B-RBC survival.[6] Three patients had HbSS and one had HbS/β⁰-thalassemia. Eleven additional study participants did not develop WAA or B-RBC antibodies.

Each patient received two or three B-RBC units in an initial transfusion episode. Each RBC unit in an episode was prepared by removing an aliquot and labelling it by incubation with biotinylating reagent. The aliquots in an episode were incubated at different reagent concentrations, yielding different biotin “densities” that were linearly related to the average numbers of biotin molecules per RBC. For each unit, the patient was transfused with the non-B-RBC portion followed by the B-RBC aliquot. After receiving the units in an episode, the patients resumed non-B-RBCs for chronic transfusion support. Blood samples were drawn at intervals of up to 18 weeks, longer than previous data collection periods of up to 8 weeks. Flow cytometry was used to determine the percentages of remaining B-RBCs of different biotin densities. Blood samples were tested weekly for four weeks for evidence of hemolysis (hemoglobin, reticulocytes, lactate dehydrogenase, direct antiglobulin test). Screening for B-RBC antibodies using gel cards was accomplished at intervals for six months.

One patient with historic Kp^a and WAA saw a reappearance of the WAA that remained during the entire study period. The second patient developed a WAA at the seventh week and reactivity to B-RBCs at week 12; both AUS persisted until the end of the study. The third patient had a history of Js^a and a WAA but only showed reactivity against B-RBC during the first three weeks of the study. The fourth patient had multiple historic alloantibodies (C, K, Fy^a, Kp^a, Go^a), historic WAA and auto-anti-D. That patient showed reactivity against B-RBC only during weeks 3-5 of the study. No patients demonstrated significant laboratory evidence of hemolysis.

Despite these patients’ alloantibodies, WAA and B-RBC antibodies, each patient’s measures of B-RBC survival, averaged for all biotin densities given to that patient, were within the ranges found in patients without antibodies. In the first patient, B-RBC survivals for the three biotin densities differed significantly; more study of this finding is needed.

Reducing a patient’s biotin dose, calculated as the sum of each biotin density multiplied by the corresponding B-RBC volume, was associated with decreased B-RBC antibody formation. This is in accordance with a prior study of autologous donors.[7]

Future B-RBC availability as a specialized laboratory test may be a useful tool in advanced patient testing to predict in vivo survival of various donor phenotypes in patients with AUS. B-RBC tests may also be helpful to aid clinicians in assessing transfusion safety in scenarios where transfusion compatibility is uncertain such as in cases of warm auto antibodies with specificities.

References:

[1] Tormey CA, Hendrickson JE. Transfusion-related red blood cell alloantibodies: induction and consequences. Blood. 2019;133(17):1821-1830.  

[2] Coleman S, Westhoff CM, Friedman DF, Chou ST. Alloimmunization in patients with sickle cell disease and under recognition of accompanying delayed hemolytic transfusion reactions. Transfusion. 2019;59(7):2282-2291. 

[3] Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff CM. High prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from Rh-matched minority donors. Blood. 2013 Aug 8;122(6):1062-71.

[4] Monocyte Monolayer Assay [Internet]. American National Red Cross [cited 2025 Feb 2]. Available from: https://www.redcrossblood.org/biomedical-services/specialty-services/laboratory-services/monocyte-monolayer-assay.html.

[5] Mock DM, Stowell SR, Franco RS, Kyosseva SV, Nalbant D, Schmidt RL, Cress GA, Strauss RG, Cancelas JA, von Goetz M, North AK, Widness JA. Antibodies against biotin-labeled red blood cells can shorten posttransfusion survival. Transfusion. 2022 Apr;62(4):770-782.

[6] Yee MEM, Zerra PE, McCoy JW, Covington ML, Stowell SR, Joiner CH, Lough CM, Delvadia BB, Josephson CD, Roback JD, Fasano RM. Post-transfusion biotin-labeled red blood cell survival studies in pediatric sickle cell disease with antibodies of uncertain significance. Transfusion. 2024 May;64(5):800-807.

[7] Gerritsma JJ, van der Bolt N, van Bruggen R, Ten Brinke A, van Dam J, Guerrero G, Vermeulen C, de Bruin S, Vlaar APJ, Biemond BJ, Nur E, van der Schoot E, Fijnvandraat K; SCORE consortium. Measurement of post-transfusion red blood cell survival kinetics in sickle cell disease and β-thalassemia: a biotin label approach. Transfusion. 2022 Oct;62(10):1984-1996.